The broad focus of this application is understanding the mechanism of action of the epidermal growth factor (EGF) receptor. For comparative and intrinsic benefit we are also studying a receptor related to the EGF receptor, the ErbB-4 receptor for heregulin. These two receptors and their ligands have significance to a variety of human cancers and furthermore, serve as model systems to elucidate receptor tyrosine kinase regulation in general. The more specific focus of this grant is the regulatory events governed by elements within the carboxyterminus of the EGF receptor and, to a lesser extent, the heregulin ErbB4 receptor. The regulatory events to be explored are the interaction of signaling transduction and trafficking proteins with these receptors. A major aim is to explore mechanistic aspects of the interaction of specific molecules, i.e. Shc and phospholipase C-gamma1 (PLC-gamma1), which have multiple phosphotyrosine recognition sequences (SH2 and/or PTB domains) with the activated EGF receptor, using both in vivo and in vitro experimental approaches. Mutagenesis will be used to create mutant full- length proteins having one or two disabled SH2 or PTB domains. Analysis of these mutants will address the issue of whether single or multiple phosphotyrosine recognition sequences are essential for productive interaction with activated receptors. Also, metabolic labeling and double affinity purification of receptor:substrate complexes will be performed to quantitate the stoichiometry of these complexes and identify other proteins present in specific association complexes. The second major focus of the application is the analysis of receptor internalization and down-regulation. Within these studies, analyses of receptor interaction with the trafficking protein AP-2 will be performed. Also, the biological significance of the internalization pathway will be explored with a comparative analysis of the internalization competent EGF receptor and the internalization incompetent ErbB-4 heregulin receptor.